The NF-.kappa.B family of transcription factors are involved in the regulation of a wide variety of cellular responses. These transcription factors mediate extracellular signals that induce expression of genes which are involved in such diverse processes as cell division, inflammation, and apoptosis. See, for example, Baldwin, Annu.Rev. Immunol. 12, 141-179 (1996); Beg and Baltimore, Science 274, 782-274 (1996); Gilmore et al., Oncogene 13, 1267-1378 (1996); Mayo, et al, Science 278, 1812-1815 (1997); and Van Antwerp et al., Science 274, 787-789 (1996).
NF-.kappa.B is anchored in the cytoplasm of most non-stimulated cells by a non-covalent interaction with one of several inhibitory proteins known as I.kappa.Bs. See for example, Baeuerle and Baltimore, Science 242, 540-546 (1988). Cellular stimuli associated with immune and inflammatory responses, for example inflammatory cytokines such as tumor necrosis factor .alpha. (TNF.alpha.) or interleukin-1 (IL-1), activate NF-.kappa.B by inducing the phosphorylation of I.kappa.Bs on specific serine residues. Phosphorylation marks the I.kappa.Bs for ubiquitination and proteosome mediated degradation. The disruption, or dissociation, of I.kappa.Bs from NF-.kappa.B unmasks the NF-.kappa.B nuclear localization signal, and facilitates the nuclear translocation of active NF-.kappa.B to the nucleus, thereby upregulating NF-.kappa.B responsive target genes. See, for example, Baeuerle and Henkel, Annu. Rev. Immunol., 12, 141-179 (1994); Baldwin, Annu.Rev. Immunol., 14, 649-683 (1996); Siebenlist et al., Annu.Rev.Cell Biol. 12, 405-455 (1994); and Verma et al, Genes Dev., 9, 2723-2735 (1995). Thus, this phosphorylation of I.kappa.Bs is a key regulatory step for NF-.kappa.B mediated processes.
Phosphorylation of I.kappa.Bs on two amino proximal serine residues (for example, in the case of I.kappa.B.alpha. serines 32 and 36) has long been appreciated to be the major regulatory step in NF-.kappa.B activation. See, for example, Baldwin, Annu.Rev. Immunol., 14, 649-683 (1996), Brown et al., Science 267, 1485-1488 (1995); DiDonato et al., Mol. Cell Biol. 16, 1295-1304 (1996); Traenckner et al., EMBO J., 14, 2876-2883 (1995). As such, an important key to elucidating the mechanism of NF-.kappa.B activation, and gaining control of the immune and inflammatory responses mediated by NF-.kappa.B activation, is determining the kinases involved.
Therefore, there is a need for finding kinases that are involved in the regulation of these processes. Initial attempts to identify the responsible kinase(s) revealed a specific I.kappa.B-kinase activity in a large, around 700 kDa, cytoplasmic complex. Chen et al., Genes Dev. 9, 1586-1597 (1995). The activation of this kinase can be mediated by mitogen-activated protein kinase kinase kinase-1 (MEKK-1), although the precise mechanism has not yet been established. Lee et al., Cell 88, 213-222 (1997).
Further experiments to decipher the functional connection between TRAFs (TNF-receptor-associated factors) and NF-.kappa.B activation led to the isolation of NF-.kappa.B-inducing kinase (NIK). Lee et al., Cell 88, 213-222 (1997); and Malinin et al., Nature 385, 540-544 (1997). NIK is a serine/threonine kinase which shares homology to MEKK-1. Phosphorylation of I.kappa.B in response to TNF.alpha. requires NIK function. Lee et al., Cell 88, 213-222 (1997); and Malinin et al., Nature 385, 540-544 (1997); Song et al. Proc. Natl.Acad.Sci 94, 9792-9796 (1997). However, NIK does not directly phosphorylate NF-.kappa.B. Lee et al., Cell 88, 213-222 (1997).
Of critical importance for elucidating, and controlling, the signaling pathways that lead to NF-.kappa.B activation is the determination and characterization of kinases that directly phosphorylate I.kappa.B. The abbreviation "IKK" is used to designate an I.kappa.B kinase. Recently, an I.kappa.B kinase (IKK), designated IKK.alpha., was identified in a yeast-two-hybrid screen with NIK as bait. Regnier et al., Cell 90, 373-383 (1997). IKK.alpha. was also purified using conventional biochemical techniques and determined to be the major I.kappa.B kinase activity induced by TNF stimulation of HeLa cells. DiDonato et al., Nature 388, 548-554 (1997). IKK.alpha. had been cloned previously in a reverse transcriptase polymerase chain reaction (RT-PCR) based search for myc-like genes containing helix-loop-helix domains and was termed CHUK (conserved helix-loop-helix ubiquitous kinase). Connelly and Marcu, Cellular and Molecular Biology Research 41, 537-549 (1995). CHUK was renamed IKK.alpha. when its function was discovered. Regnier et al. (1997). The identification of IKK.alpha. (CHUK) as a cytoplasmic kinase which phosphorylates I.kappa.B family members at their physiologically relevant sites and targets them for proteosome-mediated degradation was a major breakthrough.
The IKK.alpha. (CHUK) gene encodes a 745 amino-acid polypeptide (having a molecular mass of approximately 85 kDa). Murine and human IKK.alpha. (CHUK) cDNA clones were found to be almost identical. Connelly and Marcu, Cellular and Molecular Biology Research 41, 537-549 (1995). Another kinase, termed IKK.beta., homologous to IKK.alpha., has also been reported. Stancovski and Baltimore, Cell 91, 299-302 (1997); Woronicz et al., Science 278, 866-869 (1997); and Zandi et al. Cell 91, 243-252 (1997). IKK.alpha. and IKK.beta. have 52% overall similarity to each other and 65% identity in the kinase domain. Zandi et al., Cell 91, 243-252 (1997). An I.kappa.B kinase termed T2K has also been described in U.S. Pat. No. 5,776,717 to Cao.
The known I.kappa.B protein kinases generally phosphorylate I.kappa.Bs at specific serine residues. They specifically phosphorylate serines 32 and 36 of I.kappa.B.alpha.. Phosphorylation of both sites is required to efficiently target I.kappa.B.alpha. for destruction in vivo. Moreover, activation of IKK.alpha. and IKK.beta. occurs in response to NF-.kappa.B activating agents and mutant IKK.alpha. and IKK.beta. that are catalytically inactive block NF-.kappa.B stimulation by cytokines. These results highlight the important role played by I.kappa.B protein kinases in NF-.kappa.B activation processes. See Stancovski and Baltimore, Cell 91, 299-302 (1997) for a recent discussion of I.kappa.B kinases.
IKK.alpha. (CHUK) and IKK.beta. have structural motifs characteristic of the IKK-kinases. This includes an amino terminal serine-threonine kinase domain separated from a carboxyl proximal helix-loop-helix (H-L-H) domain by a leucine zipper-like amphipathic .alpha.-helix structure. These structural characteristics are unlike other kinases and the domains are thought to be involved in protein-protein interactions.
The discovery of IKK kinases will facilitate elucidation of the events triggered by the engagement of cytokine receptors which lead to the activation of the cytoplasmically anchored NF-.kappa.B transcription factors. This is of great importance because NF-.kappa.B gene regulation is involved in a host of pathological events, in addition to inflammatory processes. For example, NF-.kappa.B gene regulation has been implicated in the progression of acquired immune deficiency syndrome (AIDS), acute phase response, activation of immune and endothelial cells during toxic shock, allograft rejection, and radiation responses. Knowledge of the mechanisms of NF-.kappa.B activation will be invaluable in the development of therapeutic agents for these conditions.
Significantly, the discovery of kinases that are involved in activating NF-.kappa.B by phosphorylating I.kappa.Bs is critical for developing means for controlling cellular processes regulated by NF-.kappa.B. In particular, there is a need for inhibitors of I.kappa.B phosphorylation that can be used to control undesirable inflammation and immune responses. Protein kinases that act at the key regulatory step of NF-.kappa.B activation provide targets for the development of inhibitors of such responses. Discovery of additional kinases involved in the phosphorylation of I.kappa.Bs would aid in the rational development of means for controlling cellular processes regulated by the NF-.kappa.B system. Thus, there is a great need for the identification and characterizing of kinases that phosphorylate I.kappa.B.